Incorporating thioamides into proteins by native chemical ligation.

The thioamide is a versatile replacement of the peptide backbone with altered hydrogen bonding and conformational preferences, as well the ability participate in energy and electron transfer processes. Semi-synthetic incorporation of a thioamide into a protein can be used to study protein folding or protein/protein interactions using these properties. Semi-synthesis also provides the opportunity to study the role of thioamides in natural proteins. Here we outline the semi-synthesis of a model protein, the B1 domain of protein G (GB1) with a thioamide at the N-terminus or the C-terminus. The thioamide is synthetically incorporated into a fragment by solid-phase peptide synthesis, whereas the remainder of the protein is recombinantly expressed. Then, the two fragments are joined by native chemical ligation. The explicit protocol for GB1 synthesis is accompanied by examples of applications with GB1 and other proteins in structural biology and protein misfolding studies.

Somatostatin Receptor as a Molecular Imaging Target in Human and Canine Cushing Disease.

OBJECTIVES: Fluorescence-guided surgery may improve completeness of resection in transsphenoidal surgery for Cushing disease (CD) by enabling visualization of residual tumor tissue at the margins. In this review we discuss somatostatin receptors (SSTRs) as targets for fluorescence-guided surgery and overview existing SSTR-specific imaging agents. We also compare SSTR expression in normal pituitary and corticotrophinoma tissues from human and canine CD patients to assess canines as a translational model for CD. METHODS: A PubMed literature search was conducted for publications containing the terms canine, somatostatin receptor, Cushing's disease, and corticotroph adenoma. SSTR expression data from each study was documented as the presence or absence of expression or, when possible, the number of tumors expressing a given SSTR subtype within a group of tumors being studied. Studies that used reverse transcription polymerase chain reaction to quantify SSTR expression were selected for additional comparative analysis. RESULTS: SSTR5 is strongly expressed in human corticotroph adenomas and weakly expressed in surrounding pituitary parenchyma, a pattern not conclusively observed in canine patients. SSTR2 mRNA expression is similar in human normal pituitary and corticotrophinoma cells but may be significantly higher in canine normal pituitary tissue than in corticotroph tumoral tissue. Limited data were available on SSTR subtypes 1, 3, and 4. CONCLUSIONS: Further studies must fill the knowledge gaps related to species-specific SSTR expression, so using canine CD as a translational model may be premature. We do conclude that the expression profile of SSTR5 (i.e., high local expression in pituitary adenomas relative to normal surrounding tissues) makes SSTR5 a promising molecular target for FGS.

Rational design of thioamide peptides as selective inhibitors of cysteine protease cathepsin L.

Aberrant levels of cathepsin L (Cts L), a ubiquitously expressed endosomal cysteine protease, have been implicated in many diseases such as cancer and diabetes. Significantly, Cts L has been identified as a potential target for the treatment of COVID-19 due to its recently unveiled critical role in SARS-CoV-2 entry into the host cells. However, there are currently no clinically approved specific inhibitors of Cts L, as it is often challenging to obtain specificity against the many highly homologous cathepsin family cysteine proteases. Peptide-based agents are often promising protease inhibitors as they offer high selectivity and potency, but unfortunately are subject to degradation in vivo. Thioamide substitution, a single-atom O-to-S modification in the peptide backbone, has been shown to improve the proteolytic stability of peptides addressing this issue. Utilizing this approach, we demonstrate herein that good peptidyl substrates can be converted into sub-micromolar inhibitors of Cts L by a single thioamide substitution in the peptide backbone. We have designed and scanned several thioamide stabilized peptide scaffolds, in which one peptide, RS 1A, was stabilized against proteolysis by all five cathepsins (Cts L, Cts V, Cts K, Cts S, and Cts B) while inhibiting Cts L with >25-fold specificity against the other cathepsins. We further showed that this stabilized RS 1A peptide could inhibit Cts L in human liver carcinoma lysates (IC50 = 19 µM). Our study demonstrates that one can rationally design a stabilized, specific peptidyl protease inhibitor by strategic placement of a thioamide and reaffirms the place of this single-atom modification in the toolbox of peptide-based rational drug design.

Rosetta Machine Learning Models Accurately Classify Positional Effects of Thioamides on Proteolysis.

Thioamide substitutions of the peptide backbone have been shown to stabilize therapeutic and imaging peptides toward proteolysis. In order to rationally design thioamide modifications, we have developed a novel Rosetta custom score function to classify thioamide positional effects on proteolysis in substrates of serine and cysteine proteases. Peptides of interest were docked into proteases using the FlexPepDock application in Rosetta. Docked complexes were modified to contain thioamides parametrized through the creation of custom atom types in Rosetta based on ab intio simulations. Thioamide complexes were simulated, and the resultant structural complexes provided features for machine learning classification as the decomposed values of the Rosetta score function. An ensemble, majority voting model was developed to be a robust predictor of previously unpublished thioamide proteolysis holdout data. Theoretical control simulations with pseudo-atoms that modulate only one physical characteristic of the thioamide show differential effects on prediction accuracy by the optimized voting classification model. These pseudo-atom model simulations, as well as statistical analyses of the full thioamide simulations, implicate steric effects on peptide binding as being primarily responsible for thioamide positional effects on proteolytic resistance.

Studies of Thioamide Effects on Serine Protease Activity Enable Two-Site Stabilization of Cancer Imaging Peptides.

Thioamide substitutions in peptides can be used as fluorescence quenchers in protease sensors and as stabilizing modifications of hormone analogs. To guide these applications in the context of serine proteases, we here examine the cleavage of several model substrates, scanning a thioamide between the P3 and P3' positions, and identify perturbing positions for thioamide substitution. While all serine proteases tested were affected by P1 thioamidation, certain proteases were also significantly affected by other thioamide positions. We demonstrate how these findings can be applied by harnessing the combined P3/P1 effect of a single thioamide on kallikrein proteolysis to protect two key positions in a neuropeptide Y-based imaging probe, increasing its serum half-life to >24 h while maintaining potency for binding to Y1 receptor expressing cells. Such stabilized peptide probes could find application in imaging cell populations in animal models or even in clinical applications such as fluorescence-guided surgery.